Funds are requested to purchase a Bruker Elexsys 580 EPR spectrometer. This is a highly versatile commercial EPR machine that will be capable of both CW operation as well as a broad range of pulse experiments with ns time resolution. Options are requested that will enable saturation recovery as well as ELDOR experiments. This will be a multiuser instrument that will be housed and maintained by the Department of Chemistry. The primary user group will consist of five research groups at the University of Virginia: Profs. Bryant and Cafiso in the Department of Chemistry and Profs. Perozo, Tamm and Nakamoto in the Department of Molecular Physiology. The facility will also be available to other research groups at the University. This user group presently makes heavy use of the existing CW EPR capabilities at the University of Virginia, however, there are presently no instruments on campus or nearby that are capable of performing pulse EPR measurements. The work being carried out by most users in this group involves site-directed spin labeling to determine membrane protein structure and dynamics. One group is using paramagnetic species (such as nitroxides) to produce nuclear relaxation enhancements and determine dynamics in proteins. Compared to CW experiments, pulse EPR experiments enable a direct determination of electron spin-lattice relaxation rates and exchange between between spins. Methods to determine dipolar coupling between spins and estimate interspin distances can be extendent to much longer distances using pulse methods and do not require the acquisition of non-interacting reference spectra. In addition, the pulse experiment can eliminate problems due to orientational anisotropy in the CW experiment. This is user group will use pulse EPR to provide information on the structures and structural changes in proteins that facilitate transport across biological membranes, the structures and mechanisms of proteins that facilitate membrane attachment in cell signaling systems and the mechanisms of protein-mediated membrane fusion.